Chromatography material

ABSTRACT

A chromatography material is described for separation of nucleic acid mixtures in which a support is converted with a silanization reagent, in which the silanization reagent has a reactive group converted with silanization reagent, in which the silanization reagent has a reactive group converted with an alkyl- or dialkylamine, or contains a reactive group that can be converted with an alkyl- or dialkylamine, which is then reacted with the alkyl- or dialkylamine.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a 371 of PCT/EP95/00434 Feb. 7, 1995.

The invention concerns a chromatography material for separation ofnucleic acid mixtures, as well as a chromatographic method forseparation of nucleic acid mixtures.

Progress in biochemistry, molecular biology and human genetics and itsapplications in technology, medicine, pharmacy and genetic engineeringrequire rapid and systematic separation and isolation of nucleic acids.For example, a common problem in molecular biology is that a specificnucleic acid must be isolated from a naturally occurring mixture of ahundred or more components, which is contained in this mixture inconcentrations of less than 0.1%. The requirements on a chromatographicprocess therefore include, on the one hand, quantitative isolation ofthe nucleic acid and, on the other hand, quantitative separation of99.9% impurities in order to purify the nucleic acid as molecularspecies up to homogeneity.

The known chromatography methods, however, are not satisfactory withrespect to the attainable resolution of the nucleic acid mixture and therelated purity of the nucleic acid being isolated. In known methodscostly and cost-intensive equipment, like high-performance liquidchromatographs (HPLC) and/or ultracentrifuges, toxic substances, likephenol, chloroform or ethidium bromide, or substances that interfere insubsequent experiments, like RNAse or protease, are also frequentlyused.

A chromatographic support material from silica gel suitable forseparation of proteins, peptides and nucleic acids is described in U.S.Pat. No. 4,029,583, to which a stationary phase with groups that formanion or cation exchangers is bonded by means of a silanization reagent,these groups interacting with the substances being separated. Thesilanized silica gel is brought into contact with water, during whichthere is a hazard that the stationary phase will polymerize and thechromatography material will become unusable.

According to EP-B 0 104 210 nucleic acid mixtures can be separated intotheir components if a chromatography material whose support is firstconverted with a silanization reagent having a flexible chain group isused, which in turn is again converted to a finished chromatographymaterial by reaction with a reagent that forms an anion or cationexchanger. This known chromatography material separates nucleic acidmixtures using HPLC equipment. However, in long-chain nucleic acids theuse of HPLC can lead to damage (for example degradation) of long-chainnucleic acids due to high shear forces.

A process for separation of long-chain nucleic acids is described inEP-B 0 268 946 in which long-chain nucleic acids are fixed on a porousanion exchanger. The anion exchanger described in EP-B 0 104 210 is usedas anion exchanger. Satisfactory purity of the isolated nucleic acids,however, cannot be achieved.

A silanized chromatographic support is described in DE-A 39 35 098 inwhich the silanization reagent has at least one reactive group alreadyconverted with a primary or secondary hydroxylamine or contains areactive group that can be converted with a hydroxylamine. Separation ofnucleic acids in HPLC using a continuous salt gradient has beendemonstrated with this material (example 5).

However, the separation performance of known materials is not sufficientto solve difficult separation problems with simple chromatographicmethods that operate with a specified number of theoretical plates, likesimple column chromatography in which flow of the mobile phase isproduced by shear force, separations in spin columns (to be used in acentrifuge), in which flow of the mobile phase is produced bycentrifugal force, or batch methods in which the chromatography materialis in suspension and with avoidance of a continuous salt gradient, i.e.,using a step gradient. In particular, simple column chromatographicmethods according to the prior art often produced incomplete separationsduring isolation of DNA from cultures of transformed bacteria in whichvery limited amounts of DNA must be separated from RNA in a more thanthousand-fold excess. It is usually recommended in such cases that theRNA present in high excess be destroyed by RNAse-digestion toribonucleotides and shorter RNA fragments before chromatographicseparation.

However, this type of procedure has two critical shortcomings. In thefirst place, it is impossible to use the RNA in subsequent experimentsbecause of its destruction. On the other hand, there is a hazard ofcontamination of the laboratory, equipment and the isolated DNA itselfduring use of RNAse in the recommended concentrations of 100-400 μg/mL.Contamination with even the most limited amounts of RNAse leads to athreatening of RNA-forming subsequent experiments, like in vitrotranscription, or subsequent experiments in which RNA participates, likeNorthern hybridization and other solid phase hybridizations, like hybridselection.

On the other hand, during separation without RNAse digestioncontamination of RNA in the isolated DNA leads to hampering orprevention of subsequent experiments, like sequencing with fluorescencemarkers or with radioactive labeling, as well as all reactionsinitialized by "priming", for example all types of PCR.

The underlying task of the present invention is therefore to provide achromatography material for separation of nucleic acid mixtures withwhich the nucleic acids being isolated can be isolated in the highestpurity, fully dispensing with the use of toxic substances and RNAse, aswell as performance of separation with demanding and cost-intensiveequipment.

This task is solved with the features of the invention.

The present invention concerns a chromatography material for separationof nucleic acid mixtures in which a support is converted with asilanization reagent, which is characterized by the fact that thesilanization reagent has a reactive group converted with an alkyl- ordialkylamine, or contains a reactive group that can be converted with analkyl- or dialkylamine, which is then reacted with the alkyl- ordialkylamine.

The application discloses preferred variants of the chromatographymaterial according to the invention.

The invention also concerns a process for separation of nucleic acidmixtures with the chromatography material according to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is explained by the figures. In them

FIG. 1 shows separation of mixture components on an agarose gel usingthe chromatography material according to the invention;

FIG. 2 shows separation of the mixture components on an agarose gelusing a chromatography material according to the prior art;

FIG. 3 shows separation of the mixture components on an agarose gelusing the chromatography material of FIG. 2 and the chromatographymaterial of the invention;

FIG. 4 shows separation of the mixture components on an agarose gelusing a chromatography material according to another prior art; and

FIG. 5 shows separation of the mixture components on an agarose gelusing the chromatography material of FIG. 4 and the chromatographymaterial according to the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

It has turned out surprisingly that nucleic acid mixtures can beseparated with high efficiency with the chromatography materialaccording to the invention. Thus, for example, it is possible to isolateDNA with the highest purity, i.e., without RNA fractions, from mixturesthat contain only very limited amounts of DNA in addition to largeamounts of RNA. The use of toxic substances, like phenol, chloroform orethidium bromide can be fully dispensed with. Moreover, thechromatography material according to the invention can be used intechnically simple chromatographic methods, like simple columnchromatography, separations in spin columns or separations in batchmethods in which the chromatography material is in suspension or fixedon the reaction vessel, microtiter plates, pipette tips, agitator rodsor test strips, so that separation of nucleic acids can be carried outwithout using demanding and cost-intensive equipment, for example, HPLCor ultracentrifuging. Rapid group separation of RNA and DNA in a simplecolumn chromatography or a batch process is made possible, in particularavoiding RNAse.

Any appropriate support can be used as carrier. Silica gel, aluminumoxide, titanium dioxide, porous glass or a polymer support have provento be particularly suitable.

The support can have a particle size from 1 to 500 μm and a porediameter from 3 to 400 nm and consist of a porous or nonporous material.Supports with a particle size from 63 to 200 μm and a pore diameter from3 to 30 nm are particularly suitable, in which a pore diameter from 4 to6 nm is preferred.

Modification of the support for the chromatography material according tothe invention can occur in a one- or two-stage reaction. In theone-stage reaction the support is converted with a silanization reagentthat already contains an alkyl- or dialkylamino group. In the two-stagereaction the support is first converted with a silanization reagent thatcontains a reactive group after coupling to the support, which is madeto react in a second synthesis step with an alkyl- or dialkylamine.

The silanization reagent with which the support is converted has thegeneral formula

    R.sub.1 R.sub.2 R.sub.3 SiR.sub.4

in which

R₁ denotes an alkoxy group with 1 to 10 carbon atoms, a halogen atom ora dialkylamino group with alkyl groups having 1 to 6 carbon atoms,

R₂ and R₃ denote a hydrocarbon group with 1 to 10 carbon atoms, analkoxy group with 1 to 10 carbon atoms, a halogen atom, a dialkylaminogroup with alkyl groups having 1 to 6 carbon atoms or an alkyl groupinterrupted by at least one oxa or amino group with 4 to 20 carbonatoms, in which this group can also be mono- or multiply substitutedwith halogen atoms, cyano, nitro, amino, monoalkylamino, hydroxy or arylgroups and

R₄ in a one-stage synthesis represents a hydrocarbon group with 1 to 20carbon atoms or an alkyl group interrupted by at least one oxa or aminogroup, in which this group can be mono- or multiply substituted withhalogen atoms, cyano, amino, alkoxy, hydroxy, aryl or epoxy groups andcontains an alkyl- or dialkylamino group in which a group with theformula ##STR1## is particularly preferred, in which R₅ and R₆ denotealkyl groups with 1 to 10 carbon atoms, in which R₅ or R₆ can also be ahydrogen atom, or

R₄ in a two-stage synthesis is a hydrocarbon group with 1 to 20 carbonatoms or an alkyl group interrupted by at least one oxa or amino group,in which this group can be mono- or multiply substituted with halogenatoms, cyano, amino, alkylamino, dialkylamino, alkoxy, hydroxy, aryl orepoxy groups.

In the two-stage synthesis the flexible chain grouping R₄ that causesinteraction with the substance being separated is then modified byreaction with an alkyl- or dialkylamine to form an anion exchanger.

In a preferred variant of the invention diethylamine or dimethylamine isused as dialkylamine.

In the one-stage synthesis a silanization reagent is used in a preferredvariant in which R₅ and R₆ denote a methyl group.

In a preferred variant the reactive group converted or to be convertedwith the alkyl- or dialkylamine is the 3-glycidoxypropyl group.

Silanization of the support preferably occurs under anhydrous conditionsin order to prevent polymerization of the silanization reagent.

It is possible with the chromatography material according to theinvention to separate nucleic acid mixtures with high efficiency withoutcostly technical equipment, like HPLC, and with avoidance of addition ofRNAse. Lysates of transformed bacteria can be mentioned as examples of anucleic acid mixture to be separated, in which complete group separationinto RNA and DNA occurs. Moreover, it is possible to separatephagemid-DNA, phage-DNA, cosmid-DNA, genomic DNA, YAC-DNA, fragmentedDNA, tRNA, mRNA, hnRNA, snRNA, viral DNA or viroid DNA with thechromatography material according to the invention.

Separation of nucleic acid mixtures with the chromatography materialaccording to the invention occurs with a simple step gradient by washingof the column loaded with nucleic acid mixture and subsequent elution ofthe desired nucleic acid with an appropriate buffered salt solution.Most of the RNA is separated during binding of the DNA to thechromatography material according to the invention, whereupon anyremaining RNA is rinsed out during the washing process. Treatment withRNAse is thus unnecessary.

The chromatography material according to the invention can be marketedin different ways. For example, it is possible to offer thechromatography material according to the invention as a kit togetherwith the equipment required for chromatography. Other commercial formsare naturally included.

The invention is explained by the following examples without beinglimited to them.

EXAMPLES Example 1

100 g of silica gel with a pore diameter of 6 nm and a specific surfaceof 500 m² /g is mixed in 600 mL of dry xylene with 62 g of a silane withthe formula ##STR2## degassed by three-fold application of a vacuum andsubsequent pressurization with nitrogen and heated at 120° C. withexclusion of air and moisture for 2 hours. The modified silica gel wasfiltered off, washed repeatedly with xylene and tetrahydrofuran and thendried at 70° C. in vacuum.

Example 2

100 g of silica gel with a pore diameter of 4 nm and a specific surfaceof 750 m² /g is mixed in 600 mL of dry xylene with 46 g of3-glycidoxypropyltrimethoxysilane and 0.1 mL triethylamine. The reactionmixture is degassed by three-fold application of a vacuum and subsequentpressurization with nitrogen and heated at 130° C. with exclusion of airand moisture for 4 hours. It is filtered off and washed with xylene andtetrahydrofuran. The modified silica gel is dried at 50° C. in vacuum.

The product is then mixed with 600 mL dioxane and 32 g diethylamine andheated for 18 hours under reflux. The product is washed with dioxane andmethanol and dried in vacuum at 70° C.

Example 3

100 g of silica gel with a pore diameter of 30 nm and a specific surfaceof 60 m² /g is converted in 600 mL with dry xylene with 7.36 g3-glycidoxypropyltrimethoxysilane, as in example 2. The driedintermediate is heated under reflux for 15 hours in 200 mL of a 5.6Msolution of diethylamine in absolute ethanol. The product is washed withmethanol and dried in vacuum at 70° C.

Example 4

200 mg of the modified end product from example 1 is filled into acommercial solid phase extraction column (polypropylene-acid with HDPEfrits) with a volume of 4 mL.

The separation performance of the material is demonstrated withreference to isolation of plasmid DNA from the cell lysate of pBR322transformed RR-I E. coli cells.

Reference is made in the methods described here to T. Maniatis, E. F.Fritsch, J. Sambrook, "Molecular cloning", Cold Spring HarbourLaboratory Press, 1989.

10 mL of an RR-I E. coli culture transformed with pBR322, which wascultured in LB-ampicillin medium to a cell density of 9.84·10⁸ cells/mL,is centrifuged at 4000 g for 10 minutes. The supernatant is carefullyremoved and the bacteria pellet resuspended in 300 μL of a solution of50 mM Tris-HCl pH 8.0 and 10 mM EDTA. The bacterial suspension is mixedwith 300 mL of a solution of 0.2M NaOH, 1% sodium dodecylsulfate andincubated for 5 minutes at room temperature. 300 mL of a solution of3.3M potassium acetate, pH 5.5, is then added. The precipitatedpotassium dodecylsulfate is separated by centrifuging at 4° C. and10,000 g. The solid phase extraction column containing the anionexchanger is conditioned by applying 1 mL of a solution of 600 mM NaCl,100 mM NaAc, pH 5.0, and 0.15% Triton X-100. The clear supernatant ofpotassium dodecylsulfate centrifuging (cleared lysate) is applied to thecolumn. The column is then washed twice with 2 mL of a solution of 800mM NaCl. 100 mM NaAc, pH 5.0. The plasmid DNA bonded to the anionexchanger is then eluted with a solution of 1250 mM NaCl Tris-HCl, pH8.5.

Both the passage of cell lysate and the two wash solutions and theeluate are precipitated in each case by addition of 0.7 volume ofisopropanol and pelletized by centrifuging for 30 minutes at 4° C. and10,000 g. The supernatant is then discarded, whereupon the nucleic acidpellets are washed in 70% ethanol and then dissolved in TE, pH 8.0.

FIG. 1 shows separation of the mixture components on 1% TAE agarose gel.The following components were applied in the individual traces:

Trace 1: 1 kb ladder (Gibco BRL) as length standard;

Trace 8: 20% of the cleared lysate applied to the column. It correspondsto the DNA/RNA mixture being separated before separation as reference.The band for the plasmid DNA is only colored and shows strong "smiling"owing to the enormous amount of RNA;

Trace 3: 20% of the passage. It corresponds to the cleared lysate afterbinding to the anion exchanger column;

Trace 4: 20% of the first wash solution;

Trace 5: 20% of the second wash solution;

Trace 6: 20% of the elution solution.

Complete separation of the nucleic acid mixture components is apparentin the eluate in trace 6. which only shows plasmid DNA withoutcontamination with RNA.

Spectrophotometric concentration determinations of RNA and plasmid DNAin the passage, wash solutions and eluate show that the test mixturebeing separated consists of 1795.6 μg RNA and 2.795 μg plasmid DNA. DNAcontained only in an amount of 0.16% in the nucleic acid mixture cantherefore be isolated quantitatively in high purity.

Comparative Example 1

A nucleic acid mixture similar to that described in example 4 isseparated with the commercially available plasmid isolation kit "QiagenPlasmid Mini Kit" from the Diagen Co. GmbH (Hilden), product no. 12123,in which processing to the cleared lysate according to the productdescription, the only exception being that no RNAse treatment occurs.

A column containing the known ion exchanger is conditioned by applying 1mL of a solution of 750 mM NaCl, 50 mM MOPS, pH 7.0, 15% ethanol, 0.15%Triton X-100.

The clear supernatant of potassium dodecylsulfate centrifuging (clearedlysate) is applied to the column. The column is then washed twice with 2mL of a solution of 1M NaCl, 50 mM MOPS, 15% ethanol, pH 7.0. Theplasmid DNA bonded to the ion exchanger is eluted with a solution of1250 mM NaCl, Tris-HCl, 15% ethanol, pH 8.5.

Both the passage of the cell lysate and the two wash solutions andeluate are precipitated in each case by addition of 0.7 volume isopropyland pelletized by centrifuging at 4° C. and 15,000 g for 30 minutes. Thesupernatant is then discarded, whereupon the nucleic acid pellets arewashed with 70% ethanol and then dissolved in TE, pH 8.0.

FIG. 2 shows separation of the mixture components on a 1% TAE agarosegel. The following components were applied in the individual traces:

Trace 1: 1 kb ladder (Gibco BRL) as length standard;

Trace 8: 20% of the cleared lysate applied to the column. It correspondsto the DNA/RNA mixture being separated before separation as reference.The band for the plasmid DNA is only weakly colored and shows strong"smiling" because of the enormous amount of RNA;

Trace 3: 20% of the passage;

Trace 4: 20% of the first wash solution;

Trace 5: 20% of the second wash solution;

Trace 6: 20% of the elution solution.

As is apparent from FIG. 2, clean separation of the mixture componentsdoes not occur. The eluate (trace 6) still contains a significant RNAfraction in addition to plasmid DNA.

Spectrophotometric studies of the eluate show that 65 μg of RNA is stillpresent in addition to the expected 3 μg of plasmid DNA.

Comparative Example 2

A second separation is conducted with the isolated eluate fromcomparative example 1 with the modified chromatography materialaccording to the invention from example 1.

The precipitated eluate obtained in comparative example 1 is dissolvedin 450 μL TE, pH 8.0 and mixed with 450 μL of a solution of 800 mM NaCl,100 mM NaAc, pH 5.0. This DNA/RNA mixture is applied in similar fashionto example 4 to a conditioned column, washed twice with 2 mL of asolution of 800 mM NaCl, 100 mM NaAc, pH 5.0, and then eluted.

Both the eluate according to comparative example 1 and the two washsolutions and eluate are precipitated in each case by addition of 0.7volume isopropyl and pelletized by centrifuging at 4° C. and 15,000 gfor 30 minutes. The supernatant is then discarded, whereupon the nucleicacid pellets are washed with 70% ethanol and then dissolved in TE, pH8.0.

FIG. 3 shows separation of the mixture components on a 1% TAE agarosegel. The following components were applied in the individual traces:

Trace 1: 1 kb ladder (Gibco BRL) as length standard;

Trace 8: 20% of the eluate according to comparative example 1 asreference;

Trace 3: 20% of the passage;

Trace 4: 20% of the first wash solution;

Trace 5: 20% of the second wash solution:

Trace 6: 20% of the elution solution.

As is apparent from FIG. 3, the eluate of separation according tocomparative example 1, which was subjected again to separation with thechromatography material according to the invention is completelyseparated into its components RNA (trace 4) and DNA (trace 6).

Comparative Example 3

A nucleic acid mixture similar to that described in example 4 isseparated with another commercially available plasmid isolation kit"Nukleobon AX Kit PC 20-1" from Macherey-Nagel GmbH & Co. (Duren),product no. 730 571, in which processing to cleared lysate occurredaccording to the product description, the only exception being that noRNAse treatment was used.

A column containing the ion exchanger is conditioned by applying 1 mL ofa solution of 900 mM KCl, 100 mM Tris/H₃ PO₄, pH 6.3, 15% ethanol.

The clear supernatant of potassium dodecylsulfate centrifuging (clearedlysate) is applied to the column. The column is then washed twice with 2mL of a solution of 1.3M KCl, 100 mM Tris/H₃ PO₄, 15% ethanol, pH 6.3.The plasmid DNA bonded to the ion exchanger is eluted with a solution of1000 mM KCl, Tris-H₃ PO₄, 15% ethanol, pH 8.5.

Both the passage of the cell lysate and the two wash solutions andeluate are precipitated in each case by addition of 0.7 volumeisopropanol and pelletized by centrifuging at 4° C. and 15,000 g for 30minutes. The supernatant is discarded, whereupon the nucleic acidpellets are washed with 70% ethanol and then dissolved in TE, pH 8.0.

FIG. 4 shows separation of the mixture components on a 1% TAE agarosegel. The following components were applied in the individual traces:

Trace 1: 1 kb ladder (Gibco BRL) as length standard;

Trace 8: 20% of the cleared lysate applied to the column. It correspondsto the DNA/RNA mixture being separated before separation as reference.The band for the plasmid DNA is only weakly colored and shows strong"smiling" because of the enormous amount of RNA;

Trace 3: 20% of the passage;

Trace 4: 20% of the first wash solution;

Trace 5: 20% of the second wash solution:

Trace 6: 20% of the elution solution.

It is apparent from FIG. 4 that clean separation of the mixturecomponents does not occur. The eluate (trace 6) still contains asignificant RNA fraction in addition to plasmid DNA.

Spectrophotometric studies of the eluate show that 15.94 μg of RNA isstill present in addition to the expected 3 μg of plasmid DNA.

Comparative Example 4

A second separation was carried out with the isolated eluate fromcomparative example 3 with the modified chromatography materialaccording to the invention from example 1.

The precipitated eluate obtained in comparative example 3 is dissolvedin 450 μL TE, pH 8.0 and mixed with 450 μL of a solution of 800 mM NaCl,100 mM NaAc, pH 5.0. This DNA/RNA mixture is applied in similar fashionto example 4 to a conditioned column, washed twice with 2 mL of asolution of 800 mM NaCl, 100 mM NaAc, pH 5.0, and then eluted.

Of the eluate according to comparative example 3 and the two washsolutions and eluate are precipitated in each case by addition of 0.7volume isopropanol and pelletized by centrifuging at 4° C. and 15,000 gfor 30 minutes. The supernatant is then discarded, whereupon the nucleicacid pellets are washed in 70% ethanol and then dissolved in TE, pH 8.0.

FIG. 5 shows separation of the mixture components on a 1% TAE agarosegel. The following components were applied in the individual traces:

Trace 1: 1 kb ladder (Gibco BRL) as length standard;

Trace 8: 20% of the eluate of a separation according to the prior artsimilar to comparative example 3 as reference;

Trace 3: 20% of the passage;

Trace 4: 20% of the first wash solution;

Trace 5: 20% of the second wash solution;

Trace 6: 20% of the elution solution.

As is apparent from FIG. 5, the eluate of separation according to theprior art (trace 8) is fully separated into its components RNA (trace 4)and DNA (trace 6) by an additional separation with the chromatographymaterial according to the invention.

We claim:
 1. A process for chromatographic separation of DNA from RNA ofa mixture wherein both RNA and DNA are present, with chromatographymaterial wherein the chromatography material comprises:a support thathas been reacted with a silanization agent selected from the groupconsisting of(1) a silanization reagent having a reactive groupconverted with an alkyl- or dialkylamine and (2) a silanization reagentthat contains a reactive group that can be converted with an alkyl- ordialkylamine, which support has thereafter been reacted with the alkyl-or dialkylamine, said support having a particle size of 1 to 500 μm anda pore diameter between 3 to 6 nm, the process comprising the stepsof:loading the mixture on the chromatography material; washing theloaded chromatographic material, and applying an eluent comprising asalt solution to the chromatographic material.
 2. The process of claim 1wherein the chromatographic separation is carried out in a simple columnchromatography, spin columns, batch method in which the chromatographymaterial is in suspension or is fixed on reaction vessels, microtiterplates, pipette tips, agitator rods or test strips.
 3. The processaccording to claim 1 wherein the chromatographic separation is carriedout in a step gradient.
 4. The process according to claim 1 wherein noRNAase is used.
 5. The process according to claim 1 wherein the supportis selected from the group consisting of silica gel, aluminum oxide,titanium dioxide, porous glass and polymeric material.
 6. The processaccording to claim 1 wherein the support has a pore diameter from 4 to 6nm.
 7. The process according to claim 1 wherein the support has aparticle size from 63 to 200 μm.
 8. The process according to claim 1wherein the reactive group converted or to be converted with the alkylor dialkylamine is the 3-glicidoxypropyl group.
 9. The process accordingto claim 1 wherein a dialkylamine is used to convert the reactive group,and wherein the dialkylamine is diethylamine or dimethylamine.
 10. Theprocess according to claim 1 wherein the support has been reacted withthe silanization agent under anhydrous conditions.